Transfection with recombinant forms of human papillomaviruses (HPVS) associated with urogenital cancer has resulted in a number of cell lines with indefinite life span. None of these cell lines is tumorigenic. A dilemma exists because of the high percentage of asymptomatic women with papillomavirus infections. Furthermore, the exact role of HPV in the multi-stage carcinogenests process is unclear. Integration of HPV DNA and the aneuploid composition of the cells suggest the need for additional cocarcinogenic agents. Thus far, chemicals chosen because they are known to transform rodent cells to tumorigenicity and those considered relevant on the basis of epidemiological studies-have failed to convert immortal cells with HPV to the malignant state. The chromosome markers obtained after treatment with diverse chemical carcinogens are unique in the sense that they often involve specific rearrangements associated with regions known to harbor oncogenes. A subgenomic region of herpes simplex 2 (HSV-2/Bgl II N) was capable of converting immortal genital epithelial cells containing C integrated HPV-16 sequences into tumorigenic squamous cell carcinoma cells. Moreover, tumor-derived cultured cells and immortal cells that had been transfected with HSV-2/Bgl II N and kept in continuous culture subsequently lost the HSV-2 sequences. A situation analogous to the "hit and run hypothesis" developed to explain tumors that arise in animals treated with HSV-2 or HSV-2 subfragments. The HSV-2/Bgl II N sequence was ineffective on normal cells. Thus, HSV-2/Bgl II N may act as a cofactor in the genesis of a carcinoma but is not required to maintain the transformed phenotype. These in vitro results are consistent with the hypothesis that the development of cervical cancer requires additional factors besides the specific HPV types commonly found in severe dysplasia or carcinoma. This two-stage model utilizing relevant human cells demonstrates that HPVs play a necessary role in cervical malignancy and provides a system for elucidating critical cellular changes associated with progression to malignancy.